Algorithm User Guide: Cytoplasmic
Use the Aperio algorithms to adjust (tune) the parameters until the quantitative results are sufficiently accurate for the
purpose for which you intend to use the algorithm. You will want to test the algorithm on a variety of images so its
performance can be evaluated across the full spectrum of expected imaging conditions. To be successful, it is usually
necessary to limit the field of application to a particular type of tissue and a specific histological preparation. A more
narrowly defined application and consistency in slide preparation generally equates to a higher probability of success in
obtaining satisfactory algorithms results.
Aperio algorithms provided by Human Tissue Resource Center:
Positive Pixel Count
Colocalization
Color Deconvolution
Nuclear Quantification
Membrane Quantification
Microvessel Analysis
Rare Event Detection
Cytoplasmic
In histology and cytology, a variety of staining methods are used to target different types of tissues and cellular structures
and for detection of specific proteins. In an H&E stain, for example, Hematoxylin preferentially stains the nucleus, while
Eosin stains both nucleus and cytoplasm. In IHC analyses, different stains mark the presence of one or more proteins
within the cell.
The cytoplasmic algorithm is set to analyze DAB staining intensity and provide the percentage of cells containing stain
within the nucleus and the cytoplasm. The intensity for the positive stain is divided into four score classes with the ability
to add additional classed (up to 10 total) and provides an H-Score, a co-localized result and a histogram for the IHC
staining.
The cytoplasmic algorithm has the ability to differentiate between staining in the two sub-cellular compartments.
Translocation between the cytoplasm and the nucleus is a common method to regulate the activity of proteins in the cell,
especially transcription factors and cell cycle proteins. Under circumstances where certain cellular pathways are
disrupted, such as cancer, sub-cellular localization of these proteins may be altered. Therefore, pathologists or researchers
often evaluate the respective localization of these proteins in the nucleus or the cytoplasm in order to tain insight into the
status of cells and tissues.
The first 6 input parameters on every macro should NOT be changed. The next 3 parameters (Classifier
Neighborhood, Classifier, and Class Lists) are Genie parameters and should be changed.
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1 – Algorithm Input Parameters
A. Input parameters for Cytoplasmic Algorithm
 Analysis Stage – From this parameter you can select Report Results when you have fine-tuned the parameters and
are ready to run the analysis. Other stages allow you to fine-tune the parameter sets listed below.

Nuclear Stain Calibration – After selecting, you see the following parameters: Nuclear Stain (Red), Nuclear
Stain (Green), and Nuclear Stain (Blue). The default values are for the hematoxylin stain. OD value, [0.0 ≤ Value
≤ 1.0].

Positive Stain Calibration – After selecting, you see the following parameters: Positive Stain (Red), Positive Stain
(Green), and Positive Stain (Blue). The default values are for the DAB stain. OD value, [0.0 ≤ Value ≤ 1.0].

3rd Stain Calibration – After selecting, you see the following parameters: 3rd Stain (Red), 3rd Stain (Green), and
3rd Stain (Blue). Together these values specify the color of a third stain, if present. Normally, these values would
be zero as we assume you are using two stains, one to identify nuclei and the other to identify cytoplasm. OD
value, [0.0 ≤ Value ≤ 1.0].

Max Cell Dimension(µm) – This sets the maximum distance between two points within a cell in microns. Setting
the maximum cell dimension ensure s that cells are only counted once they occur partially in multiple views,
eliminating view overlap.

Clear Area Intensity – The default value is 240 for images scanned by a ScanScope and defines white balance for
the image. This is the reference for OD (optical density) = 0.
Nuclear Segmentation – After selecting Nuclear Segmentation as the Analysis Stage, you can see
the following parameters. These parameters filter and remove unwanted signals by smoothing
(Averaging Radius), removing background (Nuclear Threshold Type), and removing small nuclei
(Min Nuclear Area).
 Averaging Radius (µm) – This specifies the “smoothness” of the nuclei image. The larger the radius, the more
smooth the image.
o The parameter smooths out variations in nuclei size. It corrects improper segmenting of nuclei due to
spotty staining by smoothing pixels and improves nuclear counting accuracy.

Nuclear Segmentation Factor – This parameter has a value between 0 and 1. A value of zero uses only the nuclear
stain for segmentation, while nonzero values add in proportionally more of the positive stain.
o Nuclei can stain imperfectly. This parameter specifies how much brown stain to include in the nuclei
identification. A value of zero gives the strictest definition of what will be considered nuclei by only
considering a stain vector color of blue. A value of 1 allows both blue and brown stains to be considered.
A value of 0.2 is a good compromise.

Nuclear Threshold Type – If you choose Adaptive, the algorithm automatically adjusts to variations in stain
intensity to calculate a threshold. If you choose Manual, you will specify a fixed value.
o Selecting Adaptive allows the algorithm to adjust thresholds based on the strength of the staining. The
Manual setting is not recommended because it makes the algorithm too specific for this particular slide
rather than making the algorithm useful for a variety of slides with different staining intensity.

Min Nuclear Area (µm2) – This specifies the minimum size of nuclear area, and is used to exclude small nuclei
and for declustering neighboring nuclei.
o When nuclei are clustered together, decreasing this value allows the algorithm to separate them.
Cytoplasm Segmentation – After selecting Cytoplasm Segmentation as the Analysis Stage, you can
see the following parameter.
 Cytoplasmic Distance(µm) – This defines the maximum allowable distance for cytoplasm surrounding nuclei.
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Positivity Thresholds – After selecting Positivity Thresholds as the Analysis Stage, you can see the
following parameter. The scores for average cytoplasm intensity for the selected region are
calculated based on these thresholds. For greater granularity, define additional positive
thresholds.
 (1+) Threshold – Nuclear intensity threshold for weak (1+) [0 < Value ≤ 255].

(2+) Threshold – Nuclear intensity threshold for moderate (2+) [0 < Value ≤ 255].

(3+) Threshold – Nuclear intensity threshold for strong (3+) [0 < Value ≤ 255].
2 – Algorithm Results A. Understanding the Results
 Cytoplasm: H-Score – The H Score: The score is obtained by the formula: (3 x percentage of strongly staining
cytoplasm) + (2 x percentage of moderately staining cytoplasm) + (percentage of weakly staining cytoplasm),
giving a range of 0 to 300.

Cytoplasm: Average Positive Intensity – This is the average intensity of staining in the cytoplasm (cellular
average). 0+ cells are NOT included.

Cytoplasm: Percent Positive Cells – Percent of cells stained in the cytoplasm.

Nucleus: H-Score – The H Score: The score is obtained by the formula: (3 x percentage of strongly staining
nuclei) + (2 x percentage of moderately staining nuclei) + (percentage of weakly staining nuclei), giving a range of
0 to 300.

Nucleus: Average Positive Intensity – This is the average intentisty of stiaing in the nuclei (cellular average). 0+
cells are NOT included.

Nucleus: Percent Positive Cells – Percentage of cells stained in the nuclei.

Cytoplasm: Percent (N+) – For each cytoplasm threshold defined in the algorithm, this represents the percentage
of cells having been stained in the cytoplasm.

Nucleus: Percent (N+) – For each nucleus threshold defined in the algorithm, this represents the percentage of
cells having been stained in the nuclei.

Number of Cells – Total number of cells analyzed.

Percent Colocalized – Percentage of cells having staining in both cytoplasm and nucleus.

Area of Analysis (mm2) – Area of the slide that was analyzed.

Cytoplasm Area (mm2) – Total area identified as cytoplasm.

Nuclear Area (mm2) – Total area identified as nuclei.
Note: The first section of the Layer Attributes pane displays the algorithm results; the second portion (labeled “Algorithm Inputs”) repeats the input
parameters you specified.
3 – Tuning Parameters
A. Calibrating the Nuclear Stain
 By defining the stain color vectors, you are identifying to the Cytoplasmic algorithm which color identifies which
stain.
 After selecting Nuclear Stain Calibration, you will see the following parameters: Nuclear Stain (Red, Green, and
Blue).
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




The numbers shown define the color vectors for the nuclear stain used for your slide.
o If possible, use a separate control slide for each stain you want to analyze.
Click Tune and move the tuning window to an area that mostly contains the nuclear stain.
The tuning window displays an image of the stain after separation, and the Annotations window Tuning Layer
displays the OD values measured for that stain.
To fine –tune the stain color vectors for the nuclear stain, you can enter the values given in the Annotations
Window into the Nuclear Stain Calibration parameters.
Repeat this step for your positive stain and any additional stains.
B. Segmenting Nuclei
 Select the Nuclear Segmentation. Use the tuning window to view the nuclei circled with cytoplasm stain
removed. Here is a visual check that the algorithm is successfully finding nuclei.
o See “Input Parameters” (section 1).
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C. Segmenting Cytoplasm
 Select the Segmenting Cytoplasm. This parameter defines how far from the nucleus the cytoplasm can be in
microns; yet still be reported as cytoplasm that surrounds the nucleus.
 The blue areas in the tuning window are the nuclei and the yellow areas are the cytoplasm that surround the
nuclei.
o You should adjust the Cytoplasmic Distance parameter until the yellow regions surrounding the nuclei
are the desired size.
D. Setting Positivity Thresholds
 The Number of Positive Thresholds defines how many cytoplasm scores you want to be reported. The
default thresholds are 0 (negative), 1 (weak positive), 2 (moderate positive), and 3 (strong positive).

The parameters define how much cytoplasm stain has to be present to be reported as +1, +2, and so on.
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4 – Cytoplasmic Analysis
A. Running Analysis
 In order to see the complete results from the algorithm, the Analysis Stage parameter be set on Report
Results.
B. Interpreting Data
 The dark red in the mark-up image is defined in the Annotations window as Cytoplasm Percent (3+). This
identifies areas that are intensely stained for cytoplasm.
 The results are color-coded in yellow, orange, and red. The nuclear results are color-coded in blues.

See “Algorithm Results” (section 2) for interpretation of data.
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Disclaimer: Printed copies are NOT the official document. Please see the online PDF file for the most up-to-date version. 5/7/2014 9:32 AM